The interaction between papain and two typical ionic liquids (ILs), 1-octyl-3-methylimidazolium chloride ([C(8)mim]Cl) and 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl), was investigated by using fluorescence spectroscopy technique at a pH value of 7.4. The results suggested that ILs could quench the intrinsic fluorescence of papain probably via a static quenching mechanism. The binding constants were determined by employing the fluorescence quenching method. They were very small compared with that of volatile organic solvents, indicating that only very weak interaction between ILs and papain existed. The Gibbs free energy change (∆G), enthalpy change (∆H), and entropy change (∆S) during the interaction of papain and ILs were estimated. Negative values of these parameters indicated that the interaction between ILs and papain was a spontaneous process, also implying that hydrogen bonding and van der Waals forces played important roles in the interaction processes.